lnt. 1. Radiation Oncology Biol. Phys. Vol. 4, pp. 421427 @ Pergamon Press Inc., 1978. Printed in the U.S.A.

0

Original Contribution

TOTAL BODY IRRADIATION AND SYNGENEIC MARROW TRANSPLANTATION IN AN INBRED RAT MODEL OF ACUTE MYELOGENOUS LEUKEMIA JOEL S. GREENBERGER,M.D.,? CHARLES J. LOCKWOOD, B.S.,S§ DENNIS S. FRANCE, B.A.,+ THOMAS MCGRATHS: and WILLIAM C. MOLONEY, M.D.Sy” While acute myelogenous leukemia (AML) occurs rarely in laboratory animals, over 20 model systems have been reported. One of these, AML of the inbred Wiitar/Furth Rat, has been shown to be pathophysiologically similar to human AML. Ten days after intravenous inoculation of 1.0 X 10” cells of a tissue culture grown clonal line, rats demonstrated peripheral blood leukemia, replacement of greater than 90% of the bone marrow with distinctive malignant myeloblasts and a syndrome of hypermuramidase (lysozyme) emia and muramidasuria. Total body irradiation (TBI) at 10 days after leukemia cell passage with a marrow lethal dose (95Orad, 140 rad/min, lnCs source, 663 kV) followed by intravenous inoculation of 5.0 x lO”/kg viable syngeneic bone marrow cells produced transient complete remissions. Repopulation with transplanted marrow was detected along with increasing numbers of recognizable W/Fu AML cells in peripheral blood, marrow and central nervous system. The delayed leukemia relapse in irradiated transplanted rats compared to h-radiated non-transplanted controls suggests an interaction between surviving W/Fu AML cells and transplanted marrow. This model may be of value in studies designing a therapeutic interaction against AML by donor marrow in the chemotherapy, immunotherapy, and total body irradiated patient. Rat acute myelogenous

leukemia;

Total body irradiation;

INTRODUCTION

Bone marrow transplantation.

to using TBI alone in reducing the incidence of recurrent leukemia; however, more cures have been obtained with the combinations.36 Apparent cures in the marrow transplanted patient may result from actual eradication of the last leukemic cell by immunologic responses to leukemia or transplanted marrow associated antigens, rather than the aggressive cytotoxic therapy.36 Studies to define and optimize such mechanism(s) of cure can be carried out more rapidly in experimental models: however, there have been problems in establishing a reliable and predictive model of human AML.6 At least 10 mouse AML models have been reported including 6 which arose in inbred strains (Table 1); however, the type-C RNA viral etiology of mouse leukemia makes therapy results difficult to interpret since some viruses can infect and potentially transform the transplanted marrow. In contrast, studies

Bone marrow transplantation in the patient with acute myelogenous leukemia (AML) is now an established therapeutic option which has proved to be of definite benefit to approximately 15% of these patients.3’6 The frequency of failure of this modality because of graft vs host disease and fatal sepsis can be reduced with identical twin (syngeneic) grafts;33” however, recurrent AML remains a significant source of failure in these patients. Attempts to eradicate AML prior to transplant (and simultaneously condition the patient for an allograft if an identical twin donor is not available) have included total body irradiation (TBI) alone,36 cyclophosphamide plus TBI,36 vigorous single agent or combination chemotherapy instead of TBI,‘0*30and combination chemotherapy with TBI.* At the present time, there is no statistically valid advantage to using the combination regimens compared

technical assistance. “This work was supported by the American Cancer Society, Massachusetts Division, Inc., Research Grant 1469Cl; and the Nehemias Goren Foundation. Reprint requests to: Joel S. Greenberger, M.D., Joint Center for Radiation Therapy, 50 Binney Street, Boston, MA 02115, U.S.A.

tJoint Center for Radiation Therapy, Department of Radiation Therapy, Harvard Medical School and Sidney Farber Cancer Institute. SDivision of Hematology, Peter Bent Brigham Hospital, Boston, MA 02115, U.S.A. ORecipient of a Fuller Junior Research Fellowship of the American Cancer Society. YWe thank Vincent King and Mary B. Muse for excellent 421

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1978, Vol. 4, No. 5 and NO. 6

Table 1. Animal models of acute myelogenous

Species

Leukemia cell morphologyt

In vitro cell line reported

Mouse (strain) SL (BalbxNZB)Fl Balb/ctBalb/c$ Balb/cS NIH/Swiss C”Bl$ C”Bll: Ab, sg, db RFMS

Chloroma Monocytoma Monocytoma AMML AML CML Chloroma Chloroma AML AML

Ml GPC-11 5774 WEHIMCF-6 None None R433 None None

August* Holtzman Wistar BD-1X$ Long-Evans Wistar Wistar/Furth$ WOPS Brown-NorwayS Donryu Sprague/Dawley

AMNL Chloroma Chloroma AUL Chloroma Basophilic AML AML Chloroma AML Chloroma

Dog

AMML

leukemia

Trans plantable

Ref.

Spontaneous Paraffin Paraffin Mineral-oil Soule virus Balb: virus-l Mylv-virus R-Mulv-virus Graffi-virus Radiation

+ + + + _ + + + +

21 29 28 28,39 35 19 26 22 9 27

None JC13 MIACS 1 L5222 C843 RBL-I W/Fu AML None None DBLA-6 None

Spontaneous ?jR 20-Methylcholanthrene Ethylnitrosourea Dimethylbenzanthracene B-Chloroethylamine 3-Methylcholanthrene Dimethylbenzanthracene Dimethylbenzanthracene Ethylnitrosourea “‘Actinium

+ + +

40 11,24 33,41 20 23 2 17,25 7 32,38 1 42

AMML-S

Radiation

Etiology

Rat (Strain)

promyelocyte

tAML, acute myelogenous leukemia; AMML, acute monomyeloid mononuclear (non-myeloid) leukemia; CML, chronic myelogenous leukemia; leukemia. SIndicates origin and passage in inbred strain only. with 11 reported rat AML models including 5 arising in inbred strains, failed to define a causal relationship between endogenous (vertically-transmitted) rat RNA type-C viruses and leukemia (Table 1). For this reason rat AML may be a better system for experimental therapeutics. In the present report, syngeneic bone marrow transplant after total body irradiation (TBI) of inbred Wistar/Furth rats with advanced W/Fu AML” is shown to be a potentially valuable system for such studies. METHODS

AND MATERIALS

Leukemia induction W/Fu rats (150 g) (Charles River Breeding Laboratories, Boston) were housed 2 per cage and fed standard laboratory chow. W/Fu AML Cl 2” repassaged in vivo for 2 months intraperitoneally to weanling (6-8 weeks) W/Fu rats uniformly produced leukemia and ascites in animals prior to death.13.16 Ascitic fluid samples gave leukemia cell viability of greater than 85% by trypan blue exclusion. Disseminated leukemia was induced by i.v. inoculation into the surgically exposed jugular vein of 1.0 x lo6 ascitic fluid cells in 0.5 ml of normal saline. Peripheral blood WBC counts and differential counts were performed on daily tail

+ +

+ + +

34

leukemia; AMNL, acute AUL, acute undifferentiated

vein venous blood samples. Rats were considered ready for therapy on the day of appearance of W/Fu AML blast cells representing at least 10% of the peripheral blood WBC count. This uniformly occurred at 8-12 days after inoculation.‘6 The distinctive morphologic appearance of W/Fu AML blast cells compared to normal W/Fu rat myeloblasts has been reported.“.‘“” Diffusion chambers Diffusion chambers were prepared by attaching 0.20 p porosity polycarbonate filters (Nucleopore Corp., Los Angeles, Calif.) to each side of a 13 mm diameter lucite ring (Millipore Corp., Bedford, Mass.). Each chamber was tested for leaks by blowing air into chambers suspended in sterile distilled water. Chambers were sterilized at 80°C dry heat for 24 hr and then filled through the plastic rings with 0.1 ml of each cell suspension of 5.0 x lo5 cells, sealed with plastic pegs, and placed in Hanks solution at room temperature prior to implantation. Chamber implantation and harvest Host W/Fu rats (200g) received total body irradiation 24 hr prior to diffusion chamber placement

Total body irradiation in rat AML 0 J. S.

(13’Cs source, 663 kV, dose rate 140 rad/min). Each rat was then anestetized with ether, its abdomen shaved, and 8 chambers implanted into the peritoneal cavity through a midline abdominal incision, which was then closed with 3-O chromic catgut. Chambers were transferred to freshly irradiated hosts in each of several dose groups every seven days to minimize fibrin build-up. For cell harvest, rats were again anesthetized with ether and duplicate chambers removed and freed of connective tissue and fibrin by gentle blotting with sterile gauze. For each harvest, one of the duplicate chambers was placed, unopened in 1.5 ml of a solution of 0.5% pronase-5.0% ficoll in Roswell Park Memorial Institute (RPMI) 1640 medium and agitated for 15 min prior to opening, while the other chamber was opened directly. Pronase was observed to be superior to other proteolytic enzymes tested for completeness of dissolving fibrin covering the chambers. Each chamber was harvested by puncturing one of the membranes near the ring attachment with a sterile siliconized Pasteur pipette and fluid containing cells transferred to a sterile tube. Nine separate 0.1 ml washes with RPM1 1640 medium of each chamber were pooled with each cell harvest and assayed for total cell count, and viability by trypan blue exclusion. Cells were then pelleted by centrifugation and cover slip smears prepared from cells resuspended in fresh rat serum. Preparation

of bone marrow graft recipients

Graded doses of TBI were delivered using a 13’Cs source animal irradiation unit (663 kV, 140 rad/min dose rate). Therm0 luminescent dosimetry (TLD) measurements using a rat phantom in the lucite treatment box revealed a dose homogeneity of 93% in the total body volume. Cyclophosphamide (Cytoxan) was administered intravenously to groups of etheranesthetized rats via the surgically exposed jugular vein. Extravasation of the drug during administration excluded the animal from further study. Busulfan was given by gastric tube in a solution of 0.9% NaCl. Doses of each drug were individualized to mg/kg body weight. Control groups received no drugs. Rats were observed for spontaneous hemorrhage, and clinical signs of anemia. Bone marrow donors Wistar/Furth rats (200 g) were anesthetized with ether; femurs, tibias and humeri were excised and opened by longitudinal splitting. Marrow was scraped and harvested with sterile wooden sticks and was pooled in cold (5°C) Tyrodes solution according to the method of Santos and Tutschka.3’ The yield of cells by this method was approximately: 2.8 x 10’ cells/tibia, 6.5 X lO’cells/femur, and 7.0 x IO6cells/humerus (mean

GREENBERGER

et al.

423

of 10 specimens each). Marrow in Tyrodes solution was filtered through sterile gauze into plastic centrifuge tubes and spun at 1200 rpm for 8 min. The cell pellet was resuspended in 0.9% saline, cells counted and viability measured by trypan blue dye exclusion and found to be at or above 90%. Marrow was inoculated i.v. in a volume of l-2 ml containing 5 x lo* viable cells per kg body weight of each recipient. Rats were tail bled daily for platelet counts, differential and total white blood cell counts and hematocrits. Each irradiated recipient or control rat received 200,000 units of penicillin intramuscularly every other day. Pathology

After gross pathologic examination, histopathologic sections were prepared from cervical, lumbar and thoracic spinal cord, brain, liver, spleen, bone marrow, lymph node, and local tumors of leukemic and control animals by published procedures,‘6 and stained with hematoxylin and eosin.

RESULTS Prior studies indicated that the growth to a plateau maximum cell number in diffusion chambers (DC) of W/Fu AML cells could be increased by intrathoracic inoculation of DC bearing rats with 10”’live E. co/i bacteria.‘j Since humoral factors following TBI have also been reported to stimulate growth of normal marrow and leukemia cells, the effect of total body irradiation on the growth of W/Fu AML cells and normal W/Fu rat bone marrow cells in DC’s was first tested. Diffusion chambers (DC’s) containing 5.0 x 10’ cells were implanted and harvested at several intervals following implantation as described in the Methods. As shown in Table 2, W/Fu AML cells in unirradiated rats reached a saturation density of approximately 1 x 10’ ceils per 0.1 ml volume chamber by 10 days. This growth was not appreciably affected by either 760 or 1OOOrad TBI prior to chamber implant. In contrast, normal W/Fu rat bone marrow cells failed to grow in DC’s and cell numbers decreased over the 14 day period. Normal bone marrow (NBM) cells also failed to grow following DC transfer to previously TBI treated rats (Table 2). Cells with macrophage morphology accounted for > 50% of cells in NBM chambers at 4 days and > 90% at 14 days. These results were similar to those observed with normal human marrow in DC’s in TBI treated rats.14 Thus, the data indicated that TBI had little effect on the growth of W/Fu rat NBM or W/Fu AML cells in DC’s in uivo and that under both NBM cell growth was markedly conditions, decreased compared to W/Fu AML cells. These results suggested that viable W/Fu AML cells in a leukemic rat surviving TBI and marrow transplant

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Radiation Oncology 0 Biology 0 Physics Table

May-June 1978, Vol. 4, No. 5 and No. 6

2. Effect of total body irradiation on growth of W/Fu AML cells in diffusion intraperitoneally implanted into W/Fu rats

Cells in chamber?

Total cells in chamber (X 10’) at serial time points after implant (days) 5 7 10

1

3

W/Fu AML cl2 irradiated (760 rad) (1000 rad) Control

5.0 + 0.2 5.OkO.l 5.OkO.l

11.1 f 1.2 10.5 2 1.3 10.2 2 1.5

37.0 2 6 45.3 + 10 43.0 2 8

NBM Irradiated (760 rad) (1OOOrad) Control

5.0 -C0.2 5.OkO.3 5.OkO.l

0.3 -I-0.01 0.9 f 0.04 0.9 2 0.01

1.OkO.l 0.8 f 0.2 0.7 k 0.03

8127 79k7 754

0.2 f 0.0.1 0.3 k 0.02 0.7 k 0.04

chambers

14

107 + 16 12Ok 18 101 f 17

110* 15 115k17 102-t 15

0.05 + 6.01 0.2 k 0.01 0.09 + 0.05

0.009 rf:0.001 0.01 -I-0.01 0.01 k 0.01

t2OOg inbred W/Fu rats were irradiated as described in the Methods; then, diffusion chambers containing 5.0 x 10’ W/Fu AML cl2 cells (from tissue culture) or nucleated normal bone marrow (NBM) cells from tibias and femurs were placed into each of multiple chambers. Results are expressed as the mean and standard deviation of total cell counts from at least 4 chambers from at least 2 different rats removed at each time point under each set of conditions.

would not have a further of humoral factors.

growth advantage

because

Total body irradiation and bone marrow transplant in nonleukemic WIFu: rats Groups of at least 20 rats received each of several single doses of cyclophosphamide, Busulfan, or TBI and were observed for fall in peripheral blood counts, gastrointestinal (G.I.) side effects and time of death. Parallel groups receiving each dose of drug or TBI were inoculated 24 hr later with 5.0 x lO’/kg W/Fu rat bone marrow cells. All rats receiving 275 mg/kg cyclophosphamide alone demonstrated fatal bone marrow suppression by 10 days, which was reversible in 60% of transplanted rats. Similar results were obtained with Busulfan (60 mg/kg). Autopsied rats in drug treated or irradiated but nontransplanted groups demonstrated marrow aplasia as well as severe denudation of intestinal mucosa. Lower doses of cyclophosphamide or busulfan were not 100% bone marrow lethal in non-transplanted controls. With TBI, 100% of rats receiving 950rad demonstrated fatal bone marrow suppression by 17 days, which was reversible in a greater number (75%) of marrowtransplanted animals. As with cyclophosphamide and busulfan, rats receiving lower doses of TBI and bone marrow transplant (BMT) survived at higher frequency: however, in lower dose groups 20 day survivors were also observed in nontransplanted controls (Fig. 1.). All 20 day survivors lived past 50 days. Thus, the dose of 950rad was considered the optimum bone marrow lethal single agent in W/Fu rats which could be reconstituted by BMT and this schedule was used in our first experiments with leukemic recipients.

Days after

total body irradiation

Fig. 1. Total body irradiation of healthy adult W/Fu rats. Results are presented as the mean and standard deviation of at least 15 rats in each group receiving: Cl, 800 rad TBI; ?? , 920 rad TBI; 0, 950 rad TBI; ?? , 980 rad TBI. Effect of Tl3I and marrow transplant on rats with WIFu AML Groups of at least 20 rats inoculated 10 days previously with 1.0 x lo6 W/Fu AML cells and demonstrating at least 10% W/Fu AML blast cells in peripheral randomized

blood differential cell counts were to receive TBI and BMT, TBI alone or

Total body irradiation in rat AML r

IOO75c

=

.? 50 ? i i! 25 1 m O'~. 0

-5

IO

15.-

J. S.

GREENBERGER

425

et al.

cell counts, percent blasts (Table 3) and decreasing platelets. These results were similar to previous reports of the natural history of intravenously passaged W/Fu AML’2*‘6*‘7and parallel results with other models of noninbred rat AML.‘* Leukemic rats receiving 950 rad total body irradiation demonstrated a decrease in white blood cell counts and platelet counts to nadirs at day 4 and day 7 respectively after TBI, with subsequent rapid rise in WBC counts (Fig. 2). This elevation of WBC count was attributable to peripheral blood leukemia with > 90% AML blasts by day 15 after TBI (Table 3). In contrast, rats receiving TBI followed by syngeneic BMT demonstrated a brief but clear delay in detectable leukemia recurrence. While WBC counts rose earlier in TBI-BMT rats (Fig. 2), the population of cells was composed peripheral blood, nucleated entirely of morphologically mature leukocytes for 14 days (Table 3). Furthermore, bone marrow cells removed from tibias of representative leukemicBMT-recipients at day 14 revealed lO-25% blasts (including recognizable W/Fu AML cells), and 7580% differentiating normal marrow elements, while similar preparations obtained from irradiated but nontransplanted leukemic rats showed clusters of W/Fu AML cells, macrophages, and occasional mature granulocytes in otherwise hypoplastic marrow. Increasing peripheral blood W/Fu AML cells were detected in transplanted leukemic rats after day 15 with over 20,000 cells/mm2 by day 20. Multiple transplants of marrow in other experiments with leukemic-BMT-recipients did not further delay relapse. All relapsing rats demonstrated post-mortem evidence in marrow specimens of regenerating normal marrow and W/Fu AML cells. There was a

TBI I EMT

is

0

2u

Days after total body irradiation

Fig. 2. Total body irradiation of W/Fu rats with advanced W/Fu AML. Results are presented as the mean and standard deviation of at least 15 rats in each group receiving (a), no treatment; (Cl), 950rad TBI; or (m), 950rad TBI followed 1 day later by 5.0 x lO”/kg syngeneic bone marrow cells inoculated i.v.

no treatment. As shown in Fig. 2, leukemia in untreated rats progressed to 100% fatality by 7 days after the first appearance of 10% AML blast cells in peripheral blood, with rising peripheral white blood

Table 3. Peripheral blood differential white blood cell counts in rats receiving body irradiation and marrow transplant for W/Fu AML

Days after TBI

No treatment % NL % Blasts

total

Rats with W/Fu AML receiving TBI TBI and BMT % Blasts %NL % Blasts % NL

:

1022 2724

9022 73*3

221 1+-l

98 ~1 9951

5 7 10 15 20

44*3 NS NS NS NS

66k4 NS NS NS NS

0 8k5 6027 90+4 NS

100 92k4 4026 1023 NS

l-cl

9921

1r1 0 0 0 10+2 3Ok4

9921 100 100 9E3 7024

tRats inoculated 8-12 days previously with WlFu AML and demonstrating 8-12% leukemic blast cells in peripheral blood then received TBI (950 rad) = day 0 and bone marrow transplant (BMT) on day one. Daily peripheral white blood cell differential counts are expressed as the mean and standard deviation percent of leukemic blast cells compared to normal leukocytes (NL) for at least 10 animals at each time. The total cell counts at each point are shown in Fig. 2. NS indicates no survivors at this time.

426

Radiation Oncology 0 Biology 0 Physics

100% incidence of central nervous system involvement with both cerebral meningeal and spinal cord infiltrates in grafted relapsing rats.16 Microscopic examination of intestinal mucosa in these leukemicBMT recipients demonstrated regeneration at the time of death.

DISCUSSION The present report describes preliminary experiments with syngeneic bone marrow transplant following TBI in an inbred rat mode1 of acute myelogenous leukemia. Proliferation of W/Fu AML cells in diffusion chambers in previously TBI treated rats was shown to be no different from that observed in unirradiated rats, and NBM cells failed to grow in DCs under both sets of conditions with no effect of TBI observed on the decrease in NBM cell numbers. Thus, humoral factors liberated following TBI did not further increase the growth advantage of W/Fu AML cells over W/Fu NBM cells in DCs. To prepare rats for BMT, TBI was compared to cyclophosphamide and busulfan. The greater percentage of recovery (75%) of rats receiving BMT after a lethal dose of TBI compared to cyclophosphamide or busulfan (60%) suggested that TBI would be a better single agent for evaluatiing BMT in leukemic rats. Furthermore, busulfan had been shown to be

May-June 1978, Vol. 4, No. 5 and No. 6

ineffective against large W/Fu AML tumor burdens in our drug screening experiments.‘* The reason for not obtaining 100% recovery of BMT recipients in the present studies is unknown; however, a sterile environment was not maintained and most BMT failures died of sepsis. Leukemic rats receiving a marrow lethal dose of TBI (950 rad) all showed W/Fu AML relapse. Relapse of AML at marrow lethal doses of TBI emphasizes the inadequacy of this modality in the setting of florid leukemia, and parallels clinical reports in human AML8*‘0Y36 as well as studies with experimental nonmyeloid 1eukemias.4*5 In the present studies we observed evidence of proliferating NBM in transplanted leukemic rats prior to and associated with a delay in peripheral blood and marrow leukemia relapse. This suggests that cocultivation in the irradiated marrow stroma of transplanted syngeneic marrow and surviving AML cells did not stimulate and may have retarded AML proliferation.37 Studies on the radiobiology of W/Fu AML cells compared to gastrointestinal and normal marrow stem cells- and design of methods which optimize AML cell kill by conditioning chemotherapy with concommitant immumotherapy should aid in the rapid analysis of new clinical transplant protocols for AML.

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3. Fefer, A., Buckner, C.D., Thomas, E.D., Cheever, M.A., Clift, R.A., Glucksberg, H., Neilman, P.E., Storb, R.: Cure of hematologic neoplasia with transplantation of marrow from identical twins. N. Engl. J. Med. 279: 146148, 1977. 4. Floersheim, G.L.: Treatment of Moloney lymphoma with lethal doses of dimethyl myleran combined with injections of haemopoetic cells. Lancet 1: 228-233, 1969. 5. Floersheim, G.L., Resziewicz, M.: Bone marrow transplantation after antilymphocyte serum and lethal chemotherapy. Nature 222: 854-857, 1%9. 6. Frei, E., Schabel, F.M., Goldin, A.: Comparative chemotherapy of AKR lymphoma and human hematological neoplasia. Cancer Res. 34: 184-193, 1974. 7. Gal, F., Somfai, S., Szentirmay, Z.: Transplantable myeloid rat leukemia induced by 7,12 dimethylbenz (a) anthracene. Acta Hematol. 49: 281-291, 1973. 8. Gale, R.P., Feig, S., Opelz, G., Territo, M., Young, L., Sarna, G., Fahey, J., Cline, M.J. and the UCLA Bone Marrow Transplant Team: Bone marrow transplantation in acute leukemia using intensive chemoradiotherapy (SCARI-UCLA). Transpl. Proc. 8: 611-616, 1976.

9. Graffi, A.: Chloroleukemia of mice. Ann. N.Y. Acad. Sci. 68: 540-558, 1957. 10. Graw, R.G., Jr., Lohrman, H.P., Bull, MI., Decter, J., Herzig, G.P., Bull, J.M., Leventhal, B.G., Yankee, R.A., Herzig, R.H., Kaveger, G.R.F., Bleyer, W.A., Buja, M.L., McGinness, M.H., Alter, H.J., WhangPeng, J., Gralnick, H.R., Kirkpatrick, C.H., Henderson, E.S.: Bone marrow transplantation following combination chemotherapy immunosuppression (B.A.C.T.) in patients with acute leukemia. Transpl. Proc. 6: 349-354, 1974. Il. Greenberger, J.S., Aaronson, S.A., Rosenthal, D.S., Maloney, W.C.: Continuous production of peroxidase, esterase, alkaline phosphatase and lysozyme by clones of promyelocytes. Nature 257: 143-144, 1975. 12. Greenberger, J.S., Bocaccino, C.A., Szot, S.J., Chemotherapeutic remissions in Moloney, W.C.: Wistar/Furth rat acute myelogenous leukemia: a model for human AML. Acta Hematol. 57: 233-241, 1977. 13. Greenberger, J.S., Campos-Neto, A., Parkman, R., Rosenthal, D.S., Schlossman, S.F., Moloney, W.C.: Immunologic detection of intracellular and cell surface lysozyme with human and experimental leukemic leukocytes. Clin. Zmmunol. & Zmmunopath. 8: 318-334, 1977. 14. Greenberger, J.S., Gans, P.J., King, V., Muse, M.B., Karpas, A., Maloney, W.C.: Diffusion chamber culture in irradiated rats of myeloblasts and promyelocytes from 84 untreated patients with myeloid leukemias or refractory dysmyelopoietic anemia: Comparison to

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36. Thomas, E.D., Fefer, A., Buckner, C.O., Storb, R.: Current status of bone marrow transplantation for aplastic anemia and acute leukemia. Blood, 79: 67 l-68 1, 1977. 37. Truitt, R.L., Rimm, A.A., Saltzstein, E.C., Rose, W.C., Bortin, M.M.: Graft versus leukemia for AKR sponTranspl. Proc. 8: 569-574, taneous leukemia-lymphoma. 1976.

38. Van Bekkum, D.W., Oosterom, P.V., Dicke, K.A.: In rat leukemias vitro colony formation of transplantable in comparison with human acute myeloid leukemia. Cancer Res. 36: 941-946,

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39. Warner, N.L., Moore, M.A.S., Metcalf, D.A.: Transplantable myelomonocytic leukemia in Balb/C mice: cytology, karyotype, and muramidase content. J. Nat1 Cancer Znstit. 43: 963-982,

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